The xCELLigence® RTCA Cardio instrument expands upon ACEA’s microelectronic-based cellular assay systems to enable monitoring of cardiomyocyte beating for cardiotoxicity assessment. While it is similar to our other xCELLigence® instruments in its use of noninvasive electrical impedance monitoring to asses cellular morphology change and attachment quality in a label-free and real-time manner, the Cardio model has a higher rate of data acquisition (every 12.9 milliseconds). Because cell-induced electrical impedance is dependent on cell size/shape and how strongly the cell interacts with the plate bottom, the contraction-relaxation cycle of cardiomyocytes gives rise to a distinct, rhythmic fluctuation in impedance that is readily captured on the millisecond time scale. Changes in the intensity and periodicity of this “beating” pattern can be monitored in the short (seconds) to long (days) time regimes to assess cardiomyocyte contractility and viability in the presence of different drugs. This enables the evaluation of both short-term and long-term (chronic exposure or repeat dosing) cardiotoxicity for clinical safety testing under conditions that are maximally physiologically relevant.
The xCELLigence® RTCA Cardio instrument, which analyzes cells in an electronic 96-well microtiter plate format (E-Plate® Cardio 96), is placed in a standard CO2 cell culture incubator and interfaces via a cable with analysis and control units that are housed outside the incubator. User friendly software allows for real-time control and monitoring of the instrument, and includes real-time data display and analysis functions that provide quantitative information about the health of the cells, including viability and beating activity/irregularity.
The analysis of human iPSC-derived cardiomyocytes with the xCELLigence® RTCA Cardio instrument has proven to be an extremely accurate, highly predictive in vitro assay for assessing drug-induced cardiac liability. Since 2010, more than 20 such studies have been published. The results collectively show that the xCELLigence® RTCA Cardio system addresses previously unmet needs in in vitro cardio safety screening by:
• Being a high-throughput method for detecting functional cardiotoxicity (effects on short-term and long-term cardio beating activity) and general toxicity in vitro
• Having good correlation with clinical arrhythmogenic risk
• Providing insight into compound mechanism of action
The xCELLigence® RTCA Cardio system uses non-invasive, label-free impedance monitoring to quantitatively evaluate cardiomyocyte health/function in real-time. Cardiomyocytes are seeded in a 96-well electronic microtiter plate (E-Plate® Cardio 96) that contains gold microelectrode arrays fused to the bottom of each well. Application of a low voltage (less than 20 mV) establishes an electric current between the electrodes, which is differentially modulated by the number of cells covering the electrodes, the morphology of those cells, and the strength of cell attachment (Figure 1A). Because the cardiomyocyte contraction/relaxation cycle involves substantial changes in cell morphology and adhesion, it can be dynamically monitored using impedance (Fig. 1B). The fast data acquisition rate (12.9 ms for the entire 96-well plate) of the xCELLigence® RTCA Cardio system provides high temporal resolution for viewing subtleties of the cardiomyocyte contraction/relaxation continuum. Because impedance measurement is noninvasive, the millisecond time scale data can be combined with longer-term monitoring (hours/days) to study both the short- and long-term effect of compounds on cardiomyocyte health and function.
Figure 1. Using real-time impedance monitoring to quantitatively evaluate cardiomyocyte health and function. (A) The principle of real-time impedance monitoring. The E-Plate Cardio 96 is a 96-well microtiter plate containing gold microelectrodes integrated into the bottom of each well. Application of a low-voltage creates a current between the electrodes. The presence of adherent cells on the surface of the electrodes impedes this current in a manner which is proportional to the number of cells inside the well and the morphologic and adhesive characteristics of the cells. The impedance signal (Z) is displayed as an arbitrary, unitless parameter called Cell Index, which is a ratio that compares the impedance value in the presence of cells vs. the absence of cells. (B) Because the size/shape and attachment strength of cardiomyocytes fluctuate rhythmically during the contraction/relaxation cycle, impedance is capable of tracking this process. (C) Video of beating cardiomyocytes on an E-Plate Cardio 96.
The xCELLigence® RTCA HT system can utilize up to four 384-microtiter E-Plates®. This increased throughput is tailored toward large scale screening operations. The HT system is capable of performing all xCELLigence RTCA applications, except cell migration and invasion assays (using ACEA’s CIM-Plate®) and cardio-specific assays(analyzing cardiomyocyte contractile and electrical activities). View the table to the left for applications that the RTCA HT system is compatible with.
The high-throughput GPCR and cytotoxicity screening have been the major utilization of the xCELLigence® RTCA HT system in pharmaceutical companies. The HT system is currently used for the cytotoxicity studies in the ACEA collaborations with Alberta Center for Toxicology (ACFT) and US EPA. For more information on the high-throughput GPCR assay and cytotoxicity profiling, please download the application notes listed below.
Figure 1. Zoomed in screen shot of table for recording the contents/conditions of each well in an E-Plate.
Pre-defined protocols guide you through experimental set-up and analysis in seconds.
The RTCA Cardio Software enables facile experiment setup and execution along with powerful data analysis, while still remaining efficient and intuitive. A general synopsis of how the software is used to run and analyze an experiment is shown below.
Step 1: Record Plate Layout
Using an intuitive graphical interface the contents/conditions of each well in the electronic microtiter plate (E-Plate®) are recorded (Figure 1). Information fields for the wells include parameters such as cell type, cell number, drug identity, drug concentration, etc. Table autofilling functions, similar to what are available in Excel or other spreadsheet programs, enable rapid data entry and automatic establishment of drug concentration gradients, cell number titrations, etc. Even when multiple cell types and assay conditions are being examined, it takes just minutes to record the information for every well in a plate.
Step 2: Define Data Acquisition Parameters
Using a second table the details of data acquisition are defined.
Step 3: Running the Experiment
Press “Run” and watch as impedance data is acquired in real-time for every well in the plate. Even as data is being acquired it can be viewed and graphically manipulated.
Step 4: Data Plotting and Analysis
Using an intuitive graphical interface the real-time impedance data for all the wells, or a subset of wells, from the E-Plate can be plotted (Figure 2A). Data from multiple wells can be averaged and the coefficient of variation automatically calculated and plotted. The viewing window for the x- and y-axes can be readily adjusted, and data traces can be normalized to a specific time point (immediately before drug addition, for example). By zooming in on a short time range it is possible to view the rhythmic fluctuation in impedance resulting from cardiomyocyte beating (Figure 2B). Built-in data analysis tools enable the real-time impedance traces to be interrogated for drug-induced effects on cardiomyocyte beating activity. A total of 13 different parameters, including beating rate and amplitude, can be evaluated (Figure 2C).
Figure 2. Data plotting and analysis using the RTCA Cardio Software. (A) Screen shot of data plotting/analysis window. Here all of the curves have been normalized to the time point immediately preceding drug treatment (denoted by the bold back vertical line). Error bars represent coefficients of variation. (B) Viewing cardiomyocyte contraction. By zooming in on a shorter time range (the time points inside the red box in part “A”), it is possible to view the rhythmic fluctuation of impedance resulting from cardiomyocyte beating. (C) Built in analysis tools make it possible to identify and quantify drug-induced changes in 13 different cardiomyocyte beating parameters (including rate and amplitude).